Summary
This study indicated that acridine orange, when combined with the initial fixative stabilized soluble matrix glycosaminoglycan in situ in areas where considerable glycosaminoglycan extraction is known to occur. Acridine orange was able to diffuse through bone into areas of undecalcified mineralizing cartilage and to bind with the glycosaminoglycans in these areas equally well as in growth plate cartilage matrix. Matrix staining was visible by light microscopy without further staining and was seen to vary territorially in intensity; although cellular definition was poor. This deficiency was overcome by the additional application of p phenylenediamine, which stained the cells intensely. At the ultrastructure level, glycosaminoglycan was present as electron dense structures in the cartilage matrix. Preliminary X-ray microanalysis studies confirmed that the acridine orange stained structures contain sulphur; this finding extends the use of acridine orange further to quantitative analysis of glycosaminoglycan.
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References
Engfeldt B, Hjertquist S-O (1968) Studies on the epiphisial growth zone I. The preservation of acid glycosaminoglycans in tissues in some histotechnical procedures for electron microscopy. Virchows Archiv [Cell Pathol] 1:222–229
Ledingham JM, Simpson FO (1970) Intensification of osmium staining by p-phenylenediamine: Paraffin and epon embedding; lipid granules in renal medulla. Stain Technol 45:255–260
Ledingham JM, Simpson FO (1972) The use of p-phenylenediamine in the block to enhance osmium staining for electron microscopy. Stain Technol 47:239–243
Luft JH (1971) Ruthenium red and violet II. Fine structural localization in animal tissues. Anat Rec 171:369–416
Mitchell N, Shepard N, Harrod J (1980) The use of brominated toluidine blue O in X-ray microanalysis for proteoglycan. Histochemistry. 68:245–251
Saunders AM (1964) Histochemical identification of acid mucopolysaccharides with acridine orange. J Histochem Cytochem 12:164–170
Schefield BH, Williams BR, Doty SB (1975) Alcian blue staining of cartilage for electron microscopy. Application of the critical electrolyte concentration principle. Histochem J 7:139–149
Schümmelfeder N (1958) Histochemical significance of the polychromatic fluorescence induced in tissues stained with acridine orange. J Histochem Cytochem 6:392–393
Shepard N, Mitchell N (1976a) The localization of proteoglycan by light and electron microscopy using safranin 0. A study of epiphyseal cartilage. J Ultrastruct Res 54:451–460
Shepard N, Mitchell N (1976b) Simultaneous localization of proteoglycan by light electron microscopy using toluidine blue O. A study of epiphyseal cartilage. J Histochem Cytochem 24:621–629
Shepard N, Mitchell N (1977) The use of ruthenium red and p-phenylenediamine to stain cartilage simultaneously for light and electron microscopy. J Histochem Cytochem 25:1163–1168
Spurr AR (1969) A low-viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res 26:31–43
Thyberg J, Lohmander S, Friberg U (1973) Electron microscopic demonstration of proteoglycans in guinea pig epiphyseal cartilage. J Ultrastruct Res 45:407–427
Timár J, Gyapay G, Lapis K (1979) Acridine orange staining of the mammalian fibroblast cell coat. Histochemistry 64:189–193
Warshawsky H, Moore G (1967) A technique for the fixation and decalcification of rat incisors for electron microscopy. J Histochem Cytochem 15:542–549
Wezeman FH, Kuettner KE (1974) Selective histochemical staining of cartilage by Rivanol®. Anat Rec 180:481–490
Xipell JM, Gladwin RC (1972) The use of a low-viscosity epoxy resin in the preparation of undecalcified bone sections for light microscopy. J Microsc 96:125–129
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Shepard, N., Mitchell, N. Acridine orange stabilization of glycosaminoglycans in beginning endochondral ossification. Histochemistry 70, 107–114 (1981). https://doi.org/10.1007/BF00493202
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DOI: https://doi.org/10.1007/BF00493202